The renin-angiotensin (RA) system is an enzymatic cascade. The first (and rate-controlling) reaction is a renin-catalyzed cleavage of angiotensinogen (AGT) to yield the decapeptide angiotensin I (Ang I). The second step of the cascade is an angiotensin-converting enzyme (ACE)-catalyzed cleavage of Ang I to yield the octapeptide angiotensin II (Ang II). Ang II is the biologically-active component of the RA system.
The concept of a tissue-specific RA system is well established for the adrenal, kidney, and brain. See Racz et al. (1992) J. Clin. Endocrinol. Metab. 75:730-737; Tang et al. (1995) Am. J. Physiol. 268:F435-F446; and Sernia (1995) Regul. Pept. 57:1-18, respectively. In a tissue-specific RA system, all components of the system are produced within that tissue and the components function independently of the circulating RA system. This definition of an “tissue-specific RA system” is explicitly adopted herein. A long-unanswered question has been whether the placenta has a tissue-specific RA system.
It has been well established that the placenta produces substantial amounts of prorenin which could be converted to active renin. ACE is also present in placental tissue. The question, however, of whether AGT is produced by placental tissue has not yet been answered conclusively. Nor has it been established that human placental cells display a renin-angiotensinogen-specific receptor. The mRNA for AGT has been detected in placental tissue. See Sower et al. (1993) Hypertens. Preg. 12:163-171 and Cooper et al. (1999) Placenta 20:467-474. Also, cDNAs resembling those for kidney-derived AGT receptor proteins have been identified and cloned from placental-derived cDNA libraries. See U.S. Pat. No. 5,595,882, issued 21 Jan. 1997. However, neither the production of the AGT protein itself, nor the identification of a renin-angiotensinogen receptor, has been documented in native human placental cells.
The RA system plays a controlling role in the regulation of blood pressure and aqueous electrolytes in vivo. It also plays a key role in various hypertensive diseases, in congestive heart failure, and in various edematous diseases.
Renin is produced mainly in the kidneys. As noted above, renin cleaves AGT, present in the blood, kidney, and other organs, to produce Ang I. Ang I has very little bioactivity. It is the action of ACE that converts Ang I to Ang II. Ang II is the key bioactive component of the RA system. Another angiotensin, Ang III, is also produced by the human RA system. Ang III is a single amino acid residue shorter than Ang II, and exhibits bioactivity very similar to Ang II.
Significant quantities of AGT have been found in all regions of the placenta, including the amnion and chorion. Haigler et al. (1980) J. Biol. Chem. 255:1239-1241. Significant quantities of AGT are also present in amniotic fluid. Tewksbury et al. (1986) Clin. Chim. Acta. 158:7-12. However, the predominant form of AGT in all of these sites is a high molecular weight angiotensinogen (HMrA). HMrA bas been isolated from placental tissue. Tewksbury (2000) in Handbook of Physiology. Section 7: The Endocrine System. Vol III: Endocrine Regulation of Water and Electrolyte Balance, Fray & Goodman (eds) New York, Oxford University Press, pp 59-80. There are five distinct forms, all of which are multimers of the normally occurring low molecular weight angiotensinogen (LMrA). HMrA also occurs in the plasma of pregnant women, accounting for 16% of the total AGT in the latter half of pregnancy. Forty-seven percent of women who develop pregnancy induced hypertension (PIH) have significantly elevated levels of plasma HMrA. See Tewksbury et al. (1982) Hypertension 4:729-734; and Tewksbury et al. (1988) in Placental and Endometrial Proteins: Basic and Clinical Aspects, Utrecht, VSP, pp 651-654.
The most important biological activity of Ang II is its powerful vasoconstrictive effect, especially in the peripheral blood vessels. This action is critical to maintaining proper blood pressure. Arg II is also known to induce the adrenal zone glomerulosa to produce aldosterone. Ang II also is known to act on the adrenal medulla and sympathetic nerve ends to promote catecholamine secretion, vasopressin secretion, and prostaglandin E2 and I2 production. Due to its wide-ranging biological activity, investigations of pharmaceutically-active compounds that interfere with or otherwise modulate the intersection of Ang II with the rest of the RA systems have been many.
For example, “β-blockers” are a class of compounds known to inhibit renin production, renin being the rate-limiting enzyme in the cascade leading from angiotensinogen to Ang II. However, because β-blockers do not act upon a single receptor type, focus has shifted in recent years to a search for compounds that, rather than inhibiting the production of renin, inhibit the action of the renin enzyme itself. There have also been many pharmaceutical agents successfully launched that are ACE inhibitors, such as captopril (1-{(2S)-3-mercapto-2-methylpropionyl}-L-proline), enalapril ((S)-1-{N-[1-(ethoxycarbonyl)-3-phenylpropyl-L-alanyl}-L-proline-(Z)-2-butenedioate), delapril (N-{N-{(S)-1-ethoxycarbonyl-3-phenylpropyl}-L-alanyl}-N-(indal-2-yl) glycine hydrochloride) and alacepril (1-{(S)-3-acethylthio-2-methylpropanoyl}-L-propyl-L-phenylalanine). These drugs are used to treat essential and/or renovascular hypertension. Although these drugs (and many other ACE-inhibitors) are already in commercial use, they have undesirable side effects. Therefore, investigations continue into developing an Ang II receptor antagonist that more specifically suppresses only Ang II bioactivity.
High-throughput screening of candidate compounds is therefore a key concern of the pharmaceutical industry. Developing efficacious pharmaceutical agents requires a means to screen myriad candidate compounds for activity against a RA system receptor. Only those candidate compounds that pass the initial screening are advanced to far more expensive animal testing. Moreover, the initial screening approach should mimic, as closely as possible, the ultimate animal response, e.g., the human response, to the drug candidate being evaluated. Therefore, to assess the modulator activity of a human drug candidate, it is preferred to use human cells that have Ang II receptors (as opposed to using membrane fractions or cells derived from laboratory animals).